Thesis Defence: Development of Cav1.2-targeting fluorescent probes
August 25 at 9:00 am - 1:00 pm
Jiao Rui, supervised by Dr. Fred Menard, will defend their thesis titled “Development of Cav1.2-targeting fluorescent probes: From small molecule inhibitor conjugates to de novo cyclic peptide design” in partial fulfillment of the requirements for the degree of Master of Science in Chemistry.
An abstract for Jiao Rui’s thesis is included below.
Defences are open to all members of the campus community as well as the general public. Please email frederic.menard@ubc.ca to receive the Zoom link for this defence.
Abstract
The voltage-gated calcium channel Cav1.2 plays a significant role in the physiological activities of nerve cells and cardiac muscle cells. The expression and subcellular localization of Cav1.2 are important components of the pathogenesis of neurological diseases like Alzheimer’s. Currently, the research methods targeting Cav1.2 do not measure the real-time spatial distribution of calcium ion channels in living cells. This study aims to design and test chemical probes targeting Cav1.2, enabling researchers to conduct real-time fluorescence imaging of living cells and tissues. There are two main objectives in this study: The first is to verify the function of the existing probe FluoBar2, the second is to develop a new cyclic peptide probe that targets the extracellular domain of Cav1.2.
To validate small fluorescent molecule FluoBar2 (designed in our lab), an expression vector for the mCherry-Cav1.2 fusion protein was constructed and expressed it in HEK-293T, U138 and COS-7 cells. However, FluoBar2 was found to show insufficient specificity to serve as a Cav1.2 probe. Comprehensive computational modelling studies suggests that the binding site resides within the membrane bilayer, and may lead to weak association. Our protein model was used to identify whether another site/molecule could be exploited to achieve protein labelling.
A recent report of the structure of CaV1.2 bound to the snake toxin Calciseptine served as a template to create a de novo ligand. Our computation model was used to design a cyclic peptide molecule through RFdiffusion and PyRosetta. We evaluated its binding potential through molecular dynamics simulation, followed by residue modifications to attached a fluorescent tag. A extra-cellular d-lysine residue was selected to link a 5-FAM group. The cyclic peptide fluorescent probe was synthesized, and preliminary tests in cells show promising selectivity. Future work will complete the functional validation of the peptidic probe, as well as its use in biological assays.
